The present invention relates to genes which codes for polypeptide having a degrading activity of the sulfated-fucose-containing polysaccharide useful for Structural analysis of sulfated-fucose-containing polysaccharide and also for preparation of degraded products of the said polysaccharide; to a method for manufacturing the said polypeptide by means of genetic engineering; and to a polypeptide obtained by the said method.
The sulfated-fucose-containing polysaccharide derived from marine algae is a mixture of sulfated polysaccharides which is called fucoidan as a whole mainly consisting of fucose and that which contains galactose, glucuronic acid, xylose, mannose, glucose, etc. as well has been known. Types and amounts of such constituting sugars vary depending upon the type of the marine algae used as a material. For examples, there is a report where commercially available fucoidan manufactured by Sigma is divided into molecular species of as much as 13 [Carbohydrate Research, 255, 213xcx9c224 (1994)]. They are roughly classified into two types where one is that where uronic acid is not substantially contained but most of the constituting sugar is fucose and another is that where uronic acid is contained but fucose and mannose are contained as constituting sugars.
With regard to the biological activities of sulfated-fucose-containing polysaccharide, various activities such as potentiation of macrophage activity, inhibition of metastasis of cancer and anti-coagulation of blood have been reported. However, since sulfated-fucose-containing polysaccharide has several molecular species, it is necessary to investigate after separating and purifying the sulfated-fucose-containing polysaccharide for checking which molecular species have the real activity. However, in the conventional methods, it is not possible to separate sufficiently and, therefore, it is difficult to prepare a large amount of the product to be used as a pharmaceutical. In addition, sulfated-fucose-containing polysaccharide is a sulfated polysaccharide having a high molecular weight and, when it is used as a pharmaceutical as it is, there are problems in view of antigenicity, homogeneity, anticoagulant activity, etc. whereby it is said to be necessary that the sulfated-fucose-containing polysaccharide is to be degraded to some extent.
A method where sulfated-fucose-containing polysaccharide is enzymatically decomposed to prepare a low molecular products is advantageous since the reaction can be carried out under mild conditions and uniformly degraded products are obtained due to a substrate specificity of the enzyme. It has been reported already that awabi (ear shell or abalone), scallop, sea urchin, marine microbes, etc. produce the enzyme which degrades the sulfated-fucose-containing polysaccharide. However, such an enzyme is usually contained in living organisms only in small quantities and, in addition, there are plural sulfated-fucose-containing polysaccharide degrading enzymes therein whereby it is necessary to conduct various purifying steps for preparing a single enzyme. Moreover, amino acid sequences and genetic structures of those enzymes have not been clarified at all.
An object of the present invention is to offer the gene which codes for polypeptide having a degrading activity of the sulfated-fucose-containing polysaccharide which is useful for preparation and structural analysis of sulfated-fucose-containing polysaccharide and also for preparation of degraded sulfated-fucose-containing polysaccharide. It is also to offer a polypeptide being able to be manufactured by a genetic engineering means using the said gene and having a degrading activity of the sulfated-fucose-containing polysaccharide.
The present inventors have carried out an intensive investigation for the gene of microorganisms which produce the sulfated-fucose-containing polysaccharide degrading enzymes in order to clarify the amino acid sequence and base sequence of the polypeptide having a degrading activity of the sulfated-fucose-containing polysaccharide. As a result, they have clarified that there are each two types of gene coding for polypeptide, having a degrading activity of the sulfated-fucose-containing polysaccharide, derived from bacteria belonging to genus Alteromonas and Flavobacterium, respectively, elucidated their total base sequences, firstly clarified the amino acid sequence of the said polypeptide and succeeded in developing a method for an industrially advantageous manufacture of the polypeptide having a degrading activity of the sulfated-fucose-containing polysaccharide using the said gene whereupon they have accomplished the present invention.
Outline of the present invention will be that the first feature of the present invention relates to an isolated gene having a DNA sequence coding for the polypeptide having a degrading activity of the sulfated-fucose-containing polysaccharide or having the functionally identical activity as above.
The second feature of the present invention relates to a recombinant DNA containing the gene of the first feature of the present invention.
The third feature of the present invention relates to an expression vector where microorganisms, animal cells or plant cells in which the recombinant DNA of the second feature of the present invention is inserted as host cells.
The fourth feature of the present invention relates to a transformant which is transformed by the expression vector of the third feature of the present invention.
The fifth feature of the present invention relates to a method for the manufacture of polypeptide having a degrading activity of the sulfated-fucose-containing polysaccharide or that having the functionally identical activity as that, characterized in that, the transformant according to the fourth feature of the present invention is incubated and polypeptide having a degrading activity of the sulfated-fucose-containing polysaccharide or that having the functionally identical activity as that is collected from the incubated product.
The sixth feature of the present invention relates to a polypeptide having an amino acid sequence represented by any of the SEQ ID NO:1 to NO:4 of the Sequence Listing and having a degrading activity of the sulfated-fucose-containing polysaccharide or to a polypeptide having the functionally identical activity as that.